The rapid growth of aquaculture and scarcity of conventional fish feed supplements has prompted the introduction of new sustainable supplementation sources. In this study, the potential of five strains of fungal biomass of Ascomycetes and Zygomycetes edible filamentous fungi, Aspergillus oryzae, Neurospora intermedia, Rizhopus oryzae, Monascus purpureus, and Fusarium venenatum, cultivated on vinasse, a by-product of the bioethanol industry, as alternative protein sources for fishmeal in the fish diet was evaluated. It was observed that 5% vinasse with an initial pH of 5-6.5 can support fungal biomass yields of 34.3 +/- 2.4-118.5 +/- 3.9 g DM/L for A. Oryzae, N. intermedia, and R. oryzae. High protein contents of about 44.7%, 57.6%, and 50.9% (w/w), and fat contents of 7.0%, 3.5%, and 5.5% (w/w) were obtained for A. oryzae, N. intermedia, and R. oryzae, respectively. The latter three fungi species contained noticeable amino acid contents, including promising profiles of amino acids that are highly compatible with those of fishmeal. These findings provide evidence that fungal biomasses, with their relatively high protein content, good amino acid profiles, and other essential nutrients, are a promising supplementation alternative that can be produced from low-value by-products and organic-rich waste streams like vinasse to meet the dietary protein requirements in fish feed.